A central pathway in this insulin stimulated network is the enzyme PI 3-kinase. Inhibition of this enzyme results in a blockade of almost all of insulin's metabolic actions, as well as its effects on cell growth and differentiation. Classically, PI 3-kinase is a heterodimer consisting of a regulatory subunit of 85 kDa (p85alpha) and a catalytic subunit of 110 kDa (p110alpha). Our work and that of others, however, has demonstrated that there are actually as many as seven possible regulatory subunits of PI 3-kinase, six of which are the result of three different alternative splicing events of the p85 gene. Both PI 3-kinase activity and the alternative splicing are regulated in animal models of diabetes. The major goal of this grant is to dissect the roles of the various alternatively spliced forms of PI 3-kinase in insulin action, to explore how these components interact with each other and with other signaling molecules in the different compartments within the cell and assess their alterations in pathophysiologic states. To achieve this we will: (1) Study the similarities, differences and potentially complementary roles of various isoforms of PI 3-kinase in coupling the insulin receptor to down-stream effector systems in insulin action in vivo by creation and characterization of mice in which specific isoforms of PI 3-kinase have been knocked out using both global and conditional/tissue specific strategies of gene targeting. (2) Determine the actions of different isoforms of PI 3-kinase in differential insulin signaling by transfection of standard cell lines such as NIH 3T3 and CHO cells, more insulin responsive cells such as 3T3-L1 adipocytes and L6 myotubes, as well as cells lacking specific isoforms of PI 3-kinase derived from knockout animal models. These cells will be studied before and after reconstitution with different isoforms of PI 3-kinase, and in the case of the mouse embryo fibroblast, before and after conversion to adipocyte-like cell lines with PPARgamma2. These studies will make use of both retroviral and adenoviral vectors. (3) Determine the subcellular compartmentalization, differential partnering and differences in regulation of the various PI 3-kinase isoforms in cells in culture and tissues from intact animals, including identification and characterization of potential interacting molecules of the alternatively splice forms of PI 3-kinase using the yeast 2-hybrid system. (4) Study of the effects of hormonal treatment and disease states on differential regulation of PI 3-kinase isoforms and characterize two recently cloned phosphatidylinositol phosphate 5-phosphatases and cytoplasmic extracts of diabetic animals as possible negative regulators of the PI 3-kinase system. Together these studies should provide important insights into the role of PI 3-kinase isoforms in insulin action and diabetes.